Massimo C. Fantini

Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7.

CD4+CD25+ regulatory cells are a subpopulation of T lymphocytes of thymic origin. However, recent data suggest an alternative commitment of regulatory T cells in the periphery, although the precise mechanism is unknown. In the present work, we demonstrate that TGF-β is able to induce Foxp3 expression and subsequently a regulatory phenotype in CD4+CD25− peripheral murine T cells. Similarly, TGF-β induced Foxp3 in human CD4+CD25− T cells. Moreover, we show that the inhibitory Smad7 protein that is normally induced by TGF-β and limits TGF-β signaling, is strongly down-regulated by Foxp3 at the transcriptional level. Foxp3-mediated down-regulation of Smad7 subsequently rendered CD4+CD25− T cells highly susceptible to the morphogenic and regulatory effects of TGF-β signaling via Smad3/4. In summary, we demonstrate that TGF-β induces a regulatory phenotype in CD4+CD25− T cells through the induction of Foxp3 and a positive autoregulatory loop of TGF-β signaling due to the absence of Smad7.

IL-21 Counteracts the Regulatory T Cell-Mediated Suppression of Human CD4+ T Lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4+CD25+ regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4+CD25+ T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4+CD25− T cells and counteracts the suppressive activities of CD4+CD25+ T cells on CD4+CD25− T cells without affecting the percentage of Foxp3+ cells or survival of Treg. Additionally, CD4+CD25+ T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8+CD25− T cells but does not revert the CD4+CD25+ T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4+ T cells resistant to suppression rather than inhibiting CD4+CD25+ T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4+CD25+ T cells. Data indicate that IL-21 renders human CD4+CD25− T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.

Mongersen, an Oral SMAD7 Antisense Oligonucleotide, and Crohn’s Disease

BACKGROUND Crohns disease-related inflammation is characterized by reduced activity of the immunosuppressive cytokine transforming growth factor β1 (TGF-β1) due to high levels of SMAD7, an inhibitor of TGF-β1 signaling. Preclinical studies and a phase 1 study have shown that an oral SMAD7 antisense oligonucleotide, mongersen, targets ileal and colonic SMAD7. METHODS In a double-blind, placebo-controlled, phase 2 trial, we evaluated the efficacy of mongersen for the treatment of persons with active Crohns disease. Patients were randomly assigned to receive 10, 40, or 160 mg of mongersen or placebo per day for 2 weeks. The primary outcomes were clinical remission at day 15, defined as a Crohns Disease Activity Index (CDAI) score of less than 150, with maintenance of remission for at least 2 weeks, and the safety of mongersen treatment. A secondary outcome was clinical response (defined as a reduction of 100 points or more in the CDAI score) at day 28. RESULTS The proportions of patients who reached the primary end point were 55% and 65% for the 40-mg and 160-mg mongersen groups, respectively, as compared with 10% for the placebo group (P<0.001). There was no significant difference in the percentage of participants reaching clinical remission between the 10-mg group (12%) and the placebo group. The rate of clinical response was significantly greater among patients receiving 10 mg (37%), 40 mg (58%), or 160 mg (72%) of mongersen than among those receiving placebo (17%) (P=0.04, P<0.001, and P<0.001, respectively). Most adverse events were related to complications and symptoms of Crohns disease. CONCLUSIONS We found that study participants with Crohns disease who received mongersen had significantly higher rates of remission and clinical response than those who received placebo. (Funded by Giuliani; EudraCT number, 2011-002640-27.).

Transforming growth factor beta induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis.

Background and aims: The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25− T cells by treatment with transforming growth factor β (TGF-β). Here, we analysed the in vivo function of TGF-β induced regulatory T (Ti-Treg) cells in experimental colitis. Methods: Ti-Treg cells were generated in cell culture in the presence or absence of TGF-β and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model. Results: Ti-Treg cells significantly suppressed Th1 mediated colitis on CD4+CD62L+ T cell transfer in vivo, as shown by high resolution endoscopy, histology, immunohistochemistry, and cytokine analysis. Further analysis of in vivo and in vitro expanded Ti-Treg cells showed that exogenous interleukin 2 (IL-2) was crucial for survival and expansion of these cells. Conclusion: Our data suggest that regulatory Ti-Treg cells expand by TGF-β and exogenous IL-2 derived from effector T cells at the site of inflammation. In addition to Tr1 and thymic CD4+CD25+ T cells, peripheral Ti-Treg cells emerge as a class of regulatory T cells with therapeutic potential in T cell mediated chronic intestinal inflammation.

Regulation of Gut Inflammation and Th17 Cell Response by Interleukin-21

BACKGROUND & AIMS Interleukin (IL)-21, a T-cell-derived cytokine, is overproduced in inflammatory bowel diseases (IBD), but its role in the pathogenesis of gut inflammation remains unknown. We here examined whether IL-21 is necessary for the initiation and progress of experimental colitis and whether it regulates specific pathways of inflammation. METHODS Both dextran sulfate sodium colitis and trinitrobenzene sulfonic acid-relapsing colitis were induced in wild-type and IL-21-deficient mice. CD4(+)CD25(-) T cells from wild-type and IL-21-deficient mice were differentiated in T helper cell (Th)17-polarizing conditions, with or without IL-21 or an antagonistic IL-21R/Fc. We also examined whether blockade of IL-21 by anti-IL-21 antibody reduced IL-17 in cultures of IBD lamina propria CD3(+) T lymphocytes. Cytokines were evaluated by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay. RESULTS High IL-21 was seen in wild-type mice with dextran sulfate sodium- and trinitrobenzene sulfonic acid-relapsing colitis. IL-21-deficient mice were largely protected against both colitides and were unable to up-regulate Th17-associated molecules during gut inflammation, thus suggesting a role for IL-21 in controlling Th17 cell responses. Indeed, naïve T cells from IL-21-deficient mice failed to differentiate into Th17 cells. Treatment of developing Th17 cells from wild-type mice with IL-21R/Fc reduced IL-17 production. Moreover, in the presence of transforming growth factor-beta1, exogenous IL-21 substituted for IL-6 in driving IL-17 induction. Neutralization of IL-21 reduced IL-17 secretion by IBD lamina propria lymphocytes. CONCLUSIONS These results indicate that IL-21 is a critical regulator of inflammation and Th17 cell responses in the gut.

High resolution colonoscopy in live mice

Endoscopy in humans is a powerful method for physicians to examine the gut for inflammatory or neoplastic changes. In medical and immunological research, animal models of intestinal diseases are established key tools to investigate the mucosal immune system, colitis and cancer development in the gut. Moreover, such models represent valid systems for testing of novel drugs. In the past, mice had to be killed in order to analyze colitis activity and tumor development. The following protocol describes a method to perform high resolution endoscopic monitoring of live mice. Mice developing colitis or colonic tumors are anesthetized and examined with a miniendoscope. The endoscope is introduced via the anus and the colon is carefully insufflated with an air pump. Endoscopic pictures obtained are of high quality and allow the monitoring and grading of tumors and inflammation. In addition, colonic biopsies can be taken. This protocol can be completed within 1 h.

Cutting Edge: IL-23 Cross-Regulates IL-12 Production in T Cell-Dependent Experimental Colitis

Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. Finally, in vivo blockade of IL-12 p40 in IL-23-deficient mice rescued mice from lethal colitis. Taken together, our data identify cross-regulation of IL-12 expression by IL-23 as novel key regulatory pathway during initiation of T cell dependent colitis.

Cutting Edge: Trans-Signaling via the Soluble IL-6R Abrogates the Induction of FoxP3 in Naive CD4+CD25− T Cells

Chronic inflammatory diseases may develop when regulatory T cells (Tregs) fail to control the balance between tolerance and immunity. Alternatively, activated immune cells might prevent the induction or activation of Tregs in such diseases. In this study, we demonstrate that trans-signaling into T cells via the soluble IL-6 receptor completely abrogates the de novo induction of adaptive Tregs. Mechanistically, IL-6 trans-signaling augmented the expression of the TGF-β signaling inhibitor SMAD7. Consequently, SMAD7 overexpression in T cells using newly created transgenic mice rendered CD4+CD25− T cells resistant to the induction of FoxP3. Finally, IL-6 trans-signaling inhibited Treg-mediated suppression in a murine model of colitis. In summary, IL-6 trans-signaling into T cells emerges as a key pathway for blockade of the development of adaptive Tregs and thus may play a pivotal role in shifting the balance between effector and regulatory T cell numbers in chronic inflammatory and autoimmune diseases.

In vivo imaging of colitis and colon cancer development in mice using high resolution chromoendoscopy

Background: Mouse models of colitis and cancer are indispensable for our understanding of the pathogenesis of these diseases. In the past, mice had to be sacrificed in order to analyse colitis activity and tumour development. We have developed a safe method for high resolution endoscopic monitoring of living mice. Methods: Mice developing colitis or colonic tumours were anaesthetised using avertine and repeatedly examined by endoscopy. A novel miniendoscope (1.9 mm outer diameter), denoted Coloview, was introduced via the anus and the colon was carefully insufflated with an air pump before analysis of the colonic mucosa. An extra working channel allowed the introduction of biopsy forceps or injection needles as well as surface staining with methylene blue in order to visualise the surface of the crypts and the pit pattern architecture. Results: Endoscopic pictures obtained were of high quality and allowed monitoring and grading of disease. Scoring of colitis activity as well as tumour size and growth was possible. In addition, pit pattern analysis using chromoendoscopy permitted discrimination between inflammatory and neoplastic changes. Biopsies yielded enough tissue for molecular and histopathological analyses. Conclusions: In summary, chromoendoscopy in mice allows monitoring of the development of colitis and colon cancer with high resolution. Manipulations such as local injection of reagents or taking biopsies can be performed easily.

Regulation of homeostasis and inflammation in the intestine

The gastrointestinal tract is the largest immune interface with the environment. Exposure to large numbers of dietary and microbial antigens requires complex and highly regulated immune responses by different mucosal cell types, which result in the induction and maintenance of intestinal homeostasis. Defects in this equilibrium can disrupt the homeostatic mechanisms and lead to chronic intestinal inflammation. We review the cell populations and mechanisms involved in the control of intestinal homeostasis and inflammation, focusing on inflammatory bowel diseases. We describe some aspects of gut immunity that could alter the delicate balance between inflammatory and tolerogenic responses and result in chronic gastrointestinal tract inflammation in patients.